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Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

Lassowskat I, Böttcher C, Eschen-Lippold L, Scheel D, Lee J - Front Plant Sci (2014)

Bottom Line: Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6.An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation.Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens).

View Article: PubMed Central - PubMed

Affiliation: Department of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry Halle/Saale, Germany.

ABSTRACT
Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org).

No MeSH data available.


Related in: MedlinePlus

Modulation of metabolic defense response upon phosphorylation of MPK3/6. Wild type Col-0 DD and control plants were harvested 0–36 h after DEX treatment and methanolic extracts from leaves were analyzed by UPLC/ESI-QTOF-MS in both positive and negative mode. (A) Classification of the annotated metabolites. (B) General scheme of the biosynthesis of tryptophan (Trp)-derived metabolites. Change in levels of the precursor, Trp, is shown as mean of peak area ± standard deviation (SD) of three biological replicates. Accumulation kinetics for the control plants (Col-0 KR) are in green and for Col-0 DD in red. (IAOx = indole-3-acetaldoxime; IAN = indole-3-acetonitrile). (C) Changes in the levels of indolic glucosinolate (GLS) metabolites derived from IAOx are depicted as described in (B). (D) Changes in the levels of metabolites derived from IAN, i.e., camalexin derivatives and precursors. (HC, Hydroxycamalexin; DHCA, Dihydrocamalexic acid. For other abbreviations, see Table S1). Note that for all the graphs in (B–D), the y-axis represents the relative abundance of the metabolites (peak area of the quantifier ion, Table S19) and the x-axis the time (hours, h) after DEX treatment.
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Figure 2: Modulation of metabolic defense response upon phosphorylation of MPK3/6. Wild type Col-0 DD and control plants were harvested 0–36 h after DEX treatment and methanolic extracts from leaves were analyzed by UPLC/ESI-QTOF-MS in both positive and negative mode. (A) Classification of the annotated metabolites. (B) General scheme of the biosynthesis of tryptophan (Trp)-derived metabolites. Change in levels of the precursor, Trp, is shown as mean of peak area ± standard deviation (SD) of three biological replicates. Accumulation kinetics for the control plants (Col-0 KR) are in green and for Col-0 DD in red. (IAOx = indole-3-acetaldoxime; IAN = indole-3-acetonitrile). (C) Changes in the levels of indolic glucosinolate (GLS) metabolites derived from IAOx are depicted as described in (B). (D) Changes in the levels of metabolites derived from IAN, i.e., camalexin derivatives and precursors. (HC, Hydroxycamalexin; DHCA, Dihydrocamalexic acid. For other abbreviations, see Table S1). Note that for all the graphs in (B–D), the y-axis represents the relative abundance of the metabolites (peak area of the quantifier ion, Table S19) and the x-axis the time (hours, h) after DEX treatment.

Mentions: To investigate the impact of artificial MPK3/6 activation on plant defense metabolism without any complications from pathogens, we performed non-targeted metabolite profiling for semi-polar secondary metabolites. Samples were collected 0, 4, 8, 12, 16, 20, 24 and 36 h after DEX treatment in three independent experiments. These timepoints were selected to cover the initial time period where the MPK3/6 activation are just detectable (~4–6 h) (Figure 1A) until (and after) the initiation of tissue collapse (24–48 h). Note that at 36 h, most tissues from the MKK5DD plants are partially collapsed and flaccid, but not completely dead (i.e., brown and dry, see Figure S1B). A total of 12,913 and 10,731 molecular features were extracted from LC-MS data acquired in the positive and negative ion mode, respectively. After setting an intensity filter cutoff (>214 counts) and Two-Way ANOVA (p < 0.01), 853 and 724 differential features remained, which were further subjected to metabolite annotation (for analytical data see Tables S1, S2). In combination with known leaf metabolites this resulted in a total of 113 compounds, including 24 unidentified substances (Figure 2A). Many known defense-related compounds accumulated to high levels (see Figure 2A for compound classification and Table S1 for a complete list). The most prominent and numerous are a variety of tryptophan (Trp)-derived defense metabolites (camalexin and indole glucosinolate derivatives) as well as Trp-derived indole-3-carboxylic acid derivatives (Figure 2B and Figure S2A).


Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

Lassowskat I, Böttcher C, Eschen-Lippold L, Scheel D, Lee J - Front Plant Sci (2014)

Modulation of metabolic defense response upon phosphorylation of MPK3/6. Wild type Col-0 DD and control plants were harvested 0–36 h after DEX treatment and methanolic extracts from leaves were analyzed by UPLC/ESI-QTOF-MS in both positive and negative mode. (A) Classification of the annotated metabolites. (B) General scheme of the biosynthesis of tryptophan (Trp)-derived metabolites. Change in levels of the precursor, Trp, is shown as mean of peak area ± standard deviation (SD) of three biological replicates. Accumulation kinetics for the control plants (Col-0 KR) are in green and for Col-0 DD in red. (IAOx = indole-3-acetaldoxime; IAN = indole-3-acetonitrile). (C) Changes in the levels of indolic glucosinolate (GLS) metabolites derived from IAOx are depicted as described in (B). (D) Changes in the levels of metabolites derived from IAN, i.e., camalexin derivatives and precursors. (HC, Hydroxycamalexin; DHCA, Dihydrocamalexic acid. For other abbreviations, see Table S1). Note that for all the graphs in (B–D), the y-axis represents the relative abundance of the metabolites (peak area of the quantifier ion, Table S19) and the x-axis the time (hours, h) after DEX treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Modulation of metabolic defense response upon phosphorylation of MPK3/6. Wild type Col-0 DD and control plants were harvested 0–36 h after DEX treatment and methanolic extracts from leaves were analyzed by UPLC/ESI-QTOF-MS in both positive and negative mode. (A) Classification of the annotated metabolites. (B) General scheme of the biosynthesis of tryptophan (Trp)-derived metabolites. Change in levels of the precursor, Trp, is shown as mean of peak area ± standard deviation (SD) of three biological replicates. Accumulation kinetics for the control plants (Col-0 KR) are in green and for Col-0 DD in red. (IAOx = indole-3-acetaldoxime; IAN = indole-3-acetonitrile). (C) Changes in the levels of indolic glucosinolate (GLS) metabolites derived from IAOx are depicted as described in (B). (D) Changes in the levels of metabolites derived from IAN, i.e., camalexin derivatives and precursors. (HC, Hydroxycamalexin; DHCA, Dihydrocamalexic acid. For other abbreviations, see Table S1). Note that for all the graphs in (B–D), the y-axis represents the relative abundance of the metabolites (peak area of the quantifier ion, Table S19) and the x-axis the time (hours, h) after DEX treatment.
Mentions: To investigate the impact of artificial MPK3/6 activation on plant defense metabolism without any complications from pathogens, we performed non-targeted metabolite profiling for semi-polar secondary metabolites. Samples were collected 0, 4, 8, 12, 16, 20, 24 and 36 h after DEX treatment in three independent experiments. These timepoints were selected to cover the initial time period where the MPK3/6 activation are just detectable (~4–6 h) (Figure 1A) until (and after) the initiation of tissue collapse (24–48 h). Note that at 36 h, most tissues from the MKK5DD plants are partially collapsed and flaccid, but not completely dead (i.e., brown and dry, see Figure S1B). A total of 12,913 and 10,731 molecular features were extracted from LC-MS data acquired in the positive and negative ion mode, respectively. After setting an intensity filter cutoff (>214 counts) and Two-Way ANOVA (p < 0.01), 853 and 724 differential features remained, which were further subjected to metabolite annotation (for analytical data see Tables S1, S2). In combination with known leaf metabolites this resulted in a total of 113 compounds, including 24 unidentified substances (Figure 2A). Many known defense-related compounds accumulated to high levels (see Figure 2A for compound classification and Table S1 for a complete list). The most prominent and numerous are a variety of tryptophan (Trp)-derived defense metabolites (camalexin and indole glucosinolate derivatives) as well as Trp-derived indole-3-carboxylic acid derivatives (Figure 2B and Figure S2A).

Bottom Line: Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6.An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation.Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens).

View Article: PubMed Central - PubMed

Affiliation: Department of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry Halle/Saale, Germany.

ABSTRACT
Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org).

No MeSH data available.


Related in: MedlinePlus